Abstract

Purpose: Cell lines derived from non-small cell lung cancers (NSCLCs) revealed frequent high-level gains of chromosomal DNA at 3q23-q29 when examined by comparative genomic hybridization (CGH). Within this amplicon, a minimal common region of amplification in lung tumors had been mapped to 3q26 by earlier studies. The aim of the present work was to identify specific targets of the 3q26 amplification in NSCLCs.

Experimental Design: We examined genomic alterations in 19 NSCLC cell lines (10 derived from squamous cell carcinomas and 9 from adenocarcinomas) by using CGH and fluorescence in situ hybridization. We determined amplification and expression levels of four candidates (EVI1, TERC, SNO, and PIK3CA) in those cell lines and also in 25 primary NSCLC tumors. Because the TERC gene encodes the RNA component of human telomerase, we examined telomerase activity by the telomeric repeat amplification protocol (TRAP) assay.

Results: Copy numbers of EVI1 and TERC increased more than those of SNO and PIK3CA in our panel of NSCLC cell lines. Significant correlation between amplification and expression levels was observed only for TERC (P = 0.006), however, among these four candidate genes. Expression of TERC was also up-regulated in 24 (96%) of 25 primary NSCLC tumors compared with their nontumorous counterparts (P = 0.0001), and elevated expression of TERC was associated with high levels of telomerase activity (P = 0.048).

Conclusions: TERC is a likely target of the 3q26 amplification, and, therefore, may be a useful biomarker for diagnosis and an attractive, novel target for molecular therapy of NSCLC.

INTRODUCTION

Carcinoma of the lung is the leading cause of cancer mortality among both men and women throughout the world. NSCLC,2
which accounts for 75% to 80% of all lung cancers, consists of two major subtypes, squamous cell carcinoma and adenocarcinoma. Improvement in the efficacy of therapy for patients with NSCLC is a major public-health goal, and identification of specific genetic alterations in a given tumor could suggest optimal molecular targets for clinical management of these tumors.

Accumulated evidence suggests that multiple genetic alterations occurring sequentially in a cell lineage, at nucleotide as well as at chromosome levels, underlie the carcinogenic process in solid tumors, including NSCLCs (1)
. Amplification of chromosomal DNA is one of the mechanisms capable of activating genes that contribute to the development and progression of cancer when they are overexpressed. We have detected novel regions of amplification in various types of cancer by CGH analyses and, from such amplicons, have identified a number of candidate oncogenes that were up-regulated by DNA amplification (2, 3, 4, 5, 6, 7)
. Recently, we identified SKP2 as a target of 5p13 amplification in small-cell lung cancers (8)
.

In the work reported here, we examined 19 NSCLC cell lines by CGH to explore genomic alterations that might affect the initiation and progression of this type of tumor. Among losses and gains involving several chromosomal regions, we found the most frequent HLGs, indicative of gene amplification, at 3q23-q29. Within the 3q23-q29 amplicon, a minimal common region of amplification had been mapped to 3q26 in previous studies of NSCLC (9, 10, 11, 12, 13)
, in which an amplicon at 3q26 was detected in 25% of NSCLCs examined (14)
. Gains in DNA copy number within 3q have been reported in 16.4% of solid tumors arising in 27 different types of tissue (15)
, and in 40.2% of all types of lung cancer (15)
. Other investigators have shown that a gain of 3q can serve as a marker for the transition from severe dysplasia to invasive carcinoma of the uterine cervix (16)
, and that amplification at 3q26.3 is a parameter for tumor progression and poor prognosis in patients with head-and-neck squamous cell carcinomas (17)
. Taken together, these lines of evidence strongly suggested that the 3q26 region harbors one or more target genes the amplification of which renders them oncogenic in tumors of various types.

Earlier studies in our laboratory indicated that SNO, a regulator of tumor growth factor β (TGF-β) signaling, was a probable target for amplification at 3q26 in esophageal squamous cell carcinomas (18)
. Others have proposed PIK3CA, which encodes the catalytic subunit of phosphatidylinositol 3-kinase (PI3K), as the target for the 3q26 amplicon in ovarian cancers (19)
and NSCLCs (20)
. The 3q26 region contains other candidates, however, including EVI1 and TERC(21)
. Overexpression of EVI1, resulting from rearrangement of DNA at 3q26, is associated with myeloid leukemia and myelodysplastic syndrome (22, 23)
. TERC encodes the RNA component of human telomerase, which acts as a template for the addition of telomeric repeat sequences (24)
. To identify target(s) for the 3q26 amplification in NSCLC, we investigated amplification and expression levels of four candidate genes (EVI1, TERC, SNO, and PIK3CA), and TERC emerged as a probable target.

MATERIALS AND METHODS

Cell Lines and Primary Samples.

The 19 NSCLC cell lines chosen for this study (9 from adenocarcinomas and 10 from squamous cell carcinomas) were established and described previously (Table 1)⇓
. ABC-1, VMRC-LCD, and Lc-1sq were maintained in DMEM supplemented with 10% FCS and penicillin-streptomycin (P-S). ACC-LC-73 was maintained in RPMI 1640 supplemented with 5% FCS and P-S. All of the others were maintained in RPMI 1640 supplemented with 10% FCS and P-S.

We obtained primary samples from 25 patients with NSCLC (13 adenocarcinomas and 12 squamous-cell carcinomas) at the Chiba University Hospital, Chiba, Japan. Adjacent nontumorous lung tissues from each patient were used as controls. Before initiation of the present study, informed consent was obtained in the formal style approved by ethics committees.

CGH.

CGH was performed using directly fluorochrome-conjugated DNA as described by Kallioniemi et al.(25)
, with minor modification (26)
. Briefly, genomic DNA from each NSCLC cell line was labeled with Spectrum Green-dUTP (Vysis, Chicago, lL), and reference DNA from normal peripheral blood lymphocytes was labeled with Spectrum Orange-dUTP (Vysis) by nick translation. Labeled tumor and normal DNAs (250 ng each), together with 10 μg of Cot-1 DNA (Life Technologies, Inc., Gaithersburg, MD), were hybridized to normal male metaphase chromosome spreads. The slides were washed and counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). Shifts in CGH profiles were rated as gains and losses if they reached at least the 1.2 and 0.8 thresholds respectively. Overrepresentations were considered to be HLGs when the fluorescence ratio exceeded 1.5, as described elsewhere (2)
.

FISH.

We performed FISH experiments, using as probes, four BACs described previously (8, 27)
. We selected these BACs (RP11–33A1, RP11–816J6, RP11–543D10, and GS-162H01), containing respectively the EVI1, TERC, SNO, and PIK3CA genes, on the basis of their locations according to databases provided by the University of California-Santa Cruz3
and the NCBI.4
Briefly, each probe was labeled by nick-translation with biotin-16-dUTP or digoxigenin-11-dUTP (Roche Diagnostics, Tokyo, Japan) and was hybridized to metaphase chromosomes. Hybridization signals for multicolor FISH were detected as described elsewhere (27)
.

Southern- and Northern-Blot Analyses.

cDNA sequences of TERC (I.M.A.G.E. clone ID, 2727679), and PIK3CA (I.M.A.G.E. clone ID, 250142) were purchased from Incyte Genomics (Palo Alto, CA) and were used as probes for Southern and Northern blots. A cDNA clone of EVI1 was kindly provided by Dr. H. Hirai, University of Tokyo, Tokyo, Japan (28)
. For SNO, we designed specific PCR primers [forward (F), 5′-GGTGACCATGTTTCTCAGAC-3′ and reverse (R), 5′-CTGACTAAGTTGCAAGTGGC-3′] on the basis of its cDNA sequence (29)
and used the PCR product as a probe. Southern and Northern hybridization experiments were performed as described elsewhere (5)
.

Real-Time Quantitative RT-PCR.

We quantified mRNA levels of EVI1, TERC, SNO, and PIK3CA using a real-time fluorescence detection method described previously (7)
. Briefly, total RNA was isolated from primary NSCLC tumors and nontumorous lung tissues adjacent to each of the tumors, using Trizol (Invitrogen, Carlsbad, CA). To eliminate residual genomic DNA, the RNA extracts were treated with RNase-free DNaseI (Takara, Tokyo, Japan) before RT-PCR. Single-stranded cDNAs were generated using Superscript II reverse transcriptase (Invitrogen) following the manufacturer’s directions. Real-time quantitative PCR experiments were performed with an ABI Prism 7900 sequence-detection system (Applied Biosystems, Foster City, CA), using SYBR Green PCR Master Mix according to the manufacturer’s protocol. The primer sequences were as follows: for EVI1, (forward, 5′-TTCTGAAGCTGAGCTGTCTTCTTTT-3′, and reverse, 5′-ATCTGTACCTGCGATTTGGACTTT-3′); for TERC, (F, 5′-GTGGTGGCCATTTTTTGTCTAAC-3′, and R, 5′-TGCTCTAGAATGAACGGTGGAA-3′); for SNO, (F, 5′-TCAGCCTGATGCTCCGTGTA-3′, and R, 5′-AAGCCCCAGTGGCAAGTTC-3′); and for PIK3CA, (F, 5′-CAGGGCTTGCTGTCTCCTCTAA-3′, and R, 5′-TTTGCAGAAGACATAATTCGACACT-3′). Each primer was designed using Primer Express software (Applied Biosystems) on the basis of sequence data obtained from the NCBI database. GAPDH (Applied Biosystems) served as a reference; i.e., each sample was normalized on the basis of its GAPDH content. The thermal cycling conditions were as follows: 10 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Duplicate PCR amplifications were performed for each sample.

Telomerase Assay.

Telomerase activity was assayed by TRAPeze Telomerase Detection kits (Intergen, Purchase, NY) according to the manufacturer’s protocol. The methodology is based on a modification of the original method described by Kim et al.(30)
. Briefly, 1.0 × 106 NSCLC cells in logarithmic growth phase were lysed in 200 μl of CHAPS buffer (Intergen). After maintenance at 4°C for 30 min, the lysate was centrifuged at 12,000 × g for 20 min at 4°C. The supernatant (100 ng of protein) was subjected to the TRAP assay. After the TRAPeze reaction mixture was added, the solution was incubated at 30°C for 30 min to allow telomerase-mediated extension of a substrate oligonucleotide. The extended products were then amplified by 30 cycles of PCR (94°C for 30 s, 59°C for 30 s, and 72°C for 1 min). The PCR products were electrophoresed on 15% nondenaturing polyacrylamide gels, which were then stained with SYBR Green I (Molecular Probes, Eugene, OR). Positive enzyme activity was confirmed when a ladder of products with six-base increments starting at 50 nucleotides and a band representing the 36-bp ITAS (Intergen), were observed. The telomerase quantification control template (TSR8) from the TRAP-ese Kit was used as a positive control for estimating the telomeric repeats extended by telomerase. As a negative control, CHAPS buffer (no cellular extract) was used. The intensities of the TRAP products and ITAS bands were measured by a Bio-Imaging Analyzer (LAS 1000; Fujifilm, Tokyo, Japan) and MacBAS software (Fujifilm). The quantification of telomerase activity was calculated according to the manufacturer’s protocol as the total product generated (TPG) by formula: [Total of TRAP product ladder bands from each sample/ITAS band in each assay]/[Total of TRAP product ladder bands from TSR8/ITAS band in TSR8] × 100 = TPG (units).

Statistical Analysis.

All of the statistical analyses were performed with Stat-View 4.5 statistical software. Relationships between relative copy numbers and expression levels of each gene were determined using the Spearman’s test, with determination of correlation coefficients (r) and associated probabilities (P). The Wilcoxon signed-rank test was used to compare paired tumor and nontumor tissues for mRNA levels of each gene. The relationship between telomerase activity and mRNA levels of TERC was assessed by the Mann-Whitney test. Ps of <0.05 were considered significant.

RESULTS

Genomic Imbalances in NSCLC Cell Lines.

An overview of the genetic changes we detected among 19 NSCLC cell lines is shown in Fig. 1⇓
. All of the lines (100%) showed copy-number aberrations, and gains predominated over losses in a ratio of 1.5:1. The average number of aberrations per cell line was 16.8 (range, 7–28); average numbers of gains and losses were 10.0 (range, 6–14) and 6.8 (range, 1–14), respectively.

Summary of genetic imbalances detected by CGH in 19 NSCLC cell lines. The 22 autosomes and X chromosome are represented by ideograms showing G-banding patterns. The computerized green-to-red profiles: vertical lines on the left (red) of each chromosome ideogram, losses of genomic material; vertical lines on the right (green), copy-number gains. Yellow rectangles, HLGs. The number at the top of each vertical line, the cell line (see Table 1⇓
).

Amplification of EVI1, TERC, SNO, and PIK3CA in NSCLC Cell Lines.

We focused further examination on the common region within the 3q23-q29 amplicon that was most frequently involved in HLGs. As candidate targets for the amplicon, we selected four genes (EVI1, TERC, SNO, and PIK3CA) located at 3q26. To generate the amplicon map, we performed FISH with four BACs, each containing one of these four genes, as probes on the five NSCLC cell lines that exhibited HLGs in the 3q26 region. The precise locus of each BAC had been confirmed before the study, by FISH on normal prometaphase chromosomes (data not shown). In Lc-1sq cells we counted 16 FISH signals for EVI1 and TERC, 13 for SNO, and 10 for PIK3CA (Fig. 2, A, B, and D)⇓
. The other four cell lines (PC-10, VMRC-LCP, HUT-29, and ABC-1) showed eight, seven, six, and five FISH signals for each of the four genes, respectively (Fig. 2, A, C, and E)⇓
. These findings suggested that EVI1 and TERC lay within a more critical region of the 3q26 amplicon in NSCLC cells than did SNO and PIK3CA.

Map of the amplicon at 3q26 and representative images of two-color FISH in NSCLC cell lines. A, to the right of the chromosome 3 ideogram, the positions of four genes (EVI1, TERC, SNO, and PIK3CA) according to the University of California-Santa Cruz3 and NCBI4 databases, and the copy number per cell determined by FISH in five NSCLC cell lines. Sq, squamous cell carcinoma. Ad, adenocarcinoma. B–E, representative images of two-color FISH on metaphase chromosomes from Lc-1sq cells (B, D) and ABC-1 cells (C, E). Lc-1sq cells showed 16 FISH signals for EVI1 (B, green), 13 signals for SNO (B, red), 16 signals for TERC (D, green), and 10 signals for PIK3CA (D, red). ABC-1 cells generated five FISH signals each for EVI1 (C, green), SNO (C, red), TERC (E, green), and PIK3CA (E, red).

Relationship between Amplification and Expression Levels of Four Genes in NSCLC Cell Lines.

We examined amplification and expression levels for EVI1, TERC, SNO, and PIK3CA in our panel of 19 NSCLC cell lines by Southern and Northern blotting. All four genes exhibited amplification in multiple cell lines compared with normal genomic DNA: EVI1 in 7 (37%) of the 19 lines; TERC in 9 (47%); SNO in 7 (37%); and PIK3CA in 5 (26%; Fig. 3A⇓
). EVI1 was overexpressed in 2 (11%) of the 19 lines, TERC in 5 (26%), SNO in 5 (26%), and PIK3CA in 2 (11%; Fig. 3B⇓
). However, the expression patterns of the four genes in question did not always accord with their amplification patterns (Fig. 3)⇓
. For example, in Lc-1sq cells, TERC was overexpressed but EVI1 was not (Fig. 3B)⇓
, although both genes were amplified (Figs. 2A⇓
and 3A⇓
). Significant correlation between amplification and expression was observed only for TERC (Spearman’s rank correlation test, r = 0.647; P = 0.006; Fig. 4⇓
).

Amplification and overexpression of four genes (EVI1, TERC, SNO, and PIK3CA) lying within the 3q26 amplicon in 19 NSCLC cell lines, 9 derived from adenocarcinomas (left panels) and 10 from squamous cell carcinomas (right panels). ∗, cell lines that showed amplification and/or overexpression of these genes. A, Southern blots containing 5 μg of genomic DNA derived from NSCLC cell lines or normal peripheral lymphocytes. Additional EcoRI fragments of PIK3CA were present in some cell lines because of polymorphisms. B, Northern blot analyses using 10 μg of total RNA from each of the same NSCLC cell lines; GAPDH served as a quantity-control probe.

Correlation between amplification level (copy number) and expression level for EVI1 (A), TERC (B), SNO (C), and PIK3CA (D). From the intensity of each signal on a Southern blot, a relative copy number (X axis) was obtained that was then normalized by dividing it by the value of the normal control band after subtracting background. From the intensity of each signal on a Northern blot, a normalized expression level (Y axis) was calculated that was normalized by dividing it by the corresponding reference value obtained from GAPDH.

Expression of EVI1, TERC, SNO, and PIK3CA in Primary NSCLC Tumors.

When we determined expression levels of EVI1, TERC, SNO, and PIK3CA in paired tumor and nontumor tissues from 25 NSCLC patients using real-time quantitative RT-PCR, EVI1 was significantly overexpressed in 19 (76%) of the tumors compared with their counterpart nontumorous tissues (Wilcoxon signed-rank test, P = 0.014; Fig. 5A⇓
). Expression of TERC was even more strongly up-regulated in 24 (96%) of the 25 tumors (P = 0.0001; Fig. 5B⇓
). No expression of TERC was detectable in 22 of the 25 nontumorous tissues, although this gene was expressed in 23 of 25 tumors. SNO and PIK3CA showed higher expression in 16 (64%) and 15 (60%) of tumors, respectively, than in nontumorous tissues without statistical significance (Fig. 5, C and D)⇓
.

Relative expression of EVI1 (A), TERC (B), SNO (C), and PIK3CA (D) in 25 primary NSCLC tumors and their nontumorous counterparts. The mRNA level of each gene was evaluated by real-time quantitative RT-PCR and was normalized by expression of GAPDH.

Telomerase Activity.

Next we investigated the effect of TERC overexpression on telomerase activity in NSCLC cells using the PCR-based TRAP assay (30)
. Enzymatic activity was detected in 18 (95%) of the 19 NSCLC cell lines (Fig. 6)⇓
. To examine the relationship between telomerase activity and expression levels of TERC mRNA, the cell lines were divided into high (n = 8) and low (n = 11) groups according to the mean mRNA levels of TERC that were determined by real-time quantitative RT-PCR. Elevated expression of TERC was significantly associated with high telomerase activity (Mann-Whitney test, P = 0.048; Fig. 6B⇓
).

Association between telomerase activity and levels of TERC mRNA in NSCLC cells. A, representative images from the TRAP assay of five NSCLC cell lines, two (LK-2 and Lc-1sq) that had shown overexpression of TERC and three (HUT-15, RERF-LC-OK, and SK-LC-3) in which the gene was not overexpressed. The 36-bp ITAS is noted. B, telomerase activity in NSCLC cell lines with high versus low levels of TERC mRNA. Nineteen NSCLC cell lines were divided into high (n = 8) and low (n = 11) groups at the mean of the mRNA levels of TERC. Telomerase activity was determined as described in “Materials and Methods.” Horizontal bars, means ± SD.

DISCUSSION

Our CGH studies revealed that amplification of the 3q26 region occurred frequently in cell lines derived from NSCLCs. Results of subsequent experiments suggested that TERC was the most probable target for this amplicon among the four candidate genes examined: (a) DNA copy numbers of EVI1 and TERC increased more than those of SNO and PIK3CA in the Lc-1sq cell line (Fig. 2A)⇓
; (b) significant correlation between amplification and expression levels was observed only for TERC (Fig. 4)⇓
; (c) TERC was the gene most frequently up-regulated in primary NSCLC tumors compared with their nontumorous counterparts (Fig. 5)⇓
; and (d) elevated expression of TERC was significantly associated with high levels of telomerase activity (Fig. 6)⇓
.

Telomerase helps to stabilize the length of telomeres in human stem cells, reproductive cells, and cancer cells by adding TTAGGG repeats onto the telomeres, using its intrinsic RNA (TERC) as a template for reverse transcription (31)
. Telomerase activity has been observed in almost all human tumors, but not in normal tissues (30)
. The data shown in Fig. 5B⇓
clearly demonstrated that no detectable TERC was expressed in most of the nontumorous lung tissues adjacent to the NSCLCs we examined, but its expression was common in primary tumors, suggesting that TERC could be a useful biomarker for the diagnosis of NSCLC. Expression of an antisense TERC in human cell lines represses telomerase activity, shortens telomeres, and eventually leads to cell crisis (24)
. Together, these observations support the idea that TERC would be an attractive, novel target for molecular-based therapy for NSCLC.

Although we found a significant link between the expression level of TERC and enzymatic activity of telomerase (Fig. 6)⇓
, others have noted that TERC expression can be detected in the absence of telomerase activity and that up-regulation of TERC is an early event in carcinogenesis, whereas telomerase activity is detected only in later stages (32, 33, 34)
. Telomerase activity is regulated by multiple factors. It is known that expression levels of TERT mRNA are correlated with telomerase activity in human cancer (35)
. Therefore, we determined expression levels of TERT as well as TERC in primary NSCLC tumors. There was no significant association between expression levels of TERT and TERC (data not shown); suggesting that up-regulation of TERC might contribute to the activation of telomerase independent of TERT. Our panel of NSCLC cell lines frequently showed losses of DNA in the region harboring PINX1 (8p23; Table 2⇓
; Fig. 1⇓
); PINX1 encodes a PIN2/TRF1 binding protein that inhibits telomerase activity (36)
. These genomic changes, together with amplification of TERC at 3q26, might collaborate to activate telomerase in NSCLCs.

Earlier studies of NSCLC indicated that gains in DNA copy number at 3q26 occurred more frequently in squamous cell carcinomas than in adenocarcinomas (11, 12, 20, 37)
. Soder et al.(32)
reported that expression of TERC in NSCLC, as determined by in situ hybridization, was almost confined to squamous cell carcinoma and rare in adenocarcinoma. In our CGH studies, however, gain at 3q26 was found in 6 of the 10 cell lines derived from squamous cell carcinoma and in 6 of the 9 adenocarcinoma lines (Table 1⇓
; Fig. 1⇓
). TERC was frequently overexpressed as well as amplified in our cell lines derived from either type of tumor (Fig. 3)⇓
; moreover, we observed up-regulation of TERC expression in all of the 12 primary squamous cell carcinoma tumors that we examined, and in 12 of the 13 primary adenocarcinomas (Fig. 5)⇓
. Our results suggested that TERC was a common target of 3q26 amplification in these NSCLCs.

Because the 3q26 amplicon in NSCLCs is estimated to be more than 30 Mb long (20)
, TERC is unlikely to be the only target gene. Other candidates include BCHE(38)
, SLC2A2(38)
, EIF5A2(39)
, and SST(20)
, as well as unidentified transcripts. Activated EVI1, SNO, and/or PIK3CA may also collaborate for progression of NSCLC.

Functional studies are needed to clarify the effect of TERC on the initiation and progression of NSCLC, because TERC may become a valuable diagnostic biomarker and a novel therapeutic target for this type of lung cancer.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by: Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to J. I.), and Grants-in-Aid for Scientific Research from the Japan Society for the Program of Science (J. I., K. Y.) and from CREST of the Japan Science and Technology Corporation (J. I., K. Y.).